A solid body of basic research now makes it possible to design drugs that target the sterol biosynthesis pathway in Trypanosoma cruzi, agent of the neglected tropical disease Chagas Disease. No vaccine exists for this infection nor is one likely to become available any time soon. Meanwhile, the drugs that are available suffer from poor safety profiles, limited efficacy and emerging resistance. T. cruzi, unable to survive solely on cholesterol salvaged from the host, is vulnerable to azole inhibitors of sterol 14-demethylase (CYP51), the enzyme that is key to the parasite's ability to synthesize cholesterol endogenously. Efforts are underway to adapt the anti- fungal azoles posaconazole and ravuconazole as second-use agents for Chagas Disease, and while anti-fungal azoles do show promise, issues of safety, drug resistance and cost drive the continuing search for alternative drug chemotypes. The less than 30% sequence identity between fungal and protozoan CYP51 targets suggests that a direct development approach will be a productive route toward generating novel potent, selective and low-cost therapeutic CYP51 inhibitors. Our proposal will translate the basic science discoveries that both defined the binding mode of anti-fungal drugs to T. cruzi CYP51 and validated that enzyme as a target in the parasite into a lead optimization drug development program. We will optimize non-azole CYP51 inhibitors by generating a new library of compounds based on the LP10 lead. The medicinal chemistry effort will be aimed at optimizing pharmacokinetics, pharmacodynamics and drug safety. Structure-activity relationships derived from co-crystal structures already solved or produced with new compounds will aid lead optimization via directed chemical synthesis. This multidisciplinary program represents a two-way collaboration between the structural biology and protein crystallography expertise of Larissa Podust at the Sandler Center for Drug Discovery, UCSF, and the medicinal chemistry and drug development expertise of William Roush at Scripps Research Institute, Florida. Expertise in parasite screens and animal models of disease will be provided through the Core Laboratories of the Sandler Center directed by James McKerrow. The Roush laboratory will synthesize optimized CYP51 inhibitors from the initial leads derived in the Podust laboratory and from the screening performed in the McKerrow laboratory at UCSF. SRI International will provide pre-clinical evaluation of selected leads to aid decisions on advancement to drug candidacy. At the end of the five-year funding period we expect to file one or two new drug applications with the FDA. Thus, the short-term funding provided by the NIH is expected to have a long-term impacts on one of the most challenging and harmful diseases worldwide.